Activity and structure of EcoKMcrA.

Czapinska H, Kowalska M, Zagorskaite E, Manakova E, Slyvka A, Xu SY, Siksnys V, Sasnauskas G, Bochtler M, Nucleic Acids Res 46(18):9829-9841 (2018) Europe PMC

SASDJX2 – Modification dependent EcoKMcrA restriction endonuclease in complex with cognate hemimethylated 12bp oligoduplex

cognate hemimethylated 12-bp oligoduplex
5-methylcytosine-specific restriction enzyme A
MWI(0) 83 kDa
MWexpected 80 kDa
VPorod 69 nm3
log I(s) 4.83×103 4.83×102 4.83×101 4.83×100
cognate hemimethylated 12-bp oligoduplex 5-methylcytosine-specific restriction enzyme A small angle scattering data  s, nm-1
ln I(s)
cognate hemimethylated 12-bp oligoduplex 5-methylcytosine-specific restriction enzyme A Guinier plot ln 4.83×103 Rg: 3.9 nm 0 (3.9 nm)-2 s2
(sRg)2I(s)/I(0)
cognate hemimethylated 12-bp oligoduplex 5-methylcytosine-specific restriction enzyme A Kratky plot 1.104 0 √3 sRg
p(r)
cognate hemimethylated 12-bp oligoduplex 5-methylcytosine-specific restriction enzyme A pair distance distribution function Rg: 4.0 nm 0 Dmax: 13.5 nm

Data validation

Fits and models

log I(s)
 s, nm-1
cognate hemimethylated 12-bp oligoduplex 5-methylcytosine-specific restriction enzyme A SASREF model
log I(s)
 s, nm-1
cognate hemimethylated 12-bp oligoduplex 5-methylcytosine-specific restriction enzyme A OTHER model
log I(s)
 s, nm-1
cognate hemimethylated 12-bp oligoduplex 5-methylcytosine-specific restriction enzyme A OTHER model
Synchrotron SAXS data from solutions of EcoKMcrA restriction endonuclease bound to 12bp oligoduplex DNA in 20 mM Tris–HCl pH 7.5, 200 mM KCl, 0.1 mM EDTA, 0.01% (w/v) sodium azide and 1 mM DTT, were collected on the EMBL P12 beam line at PETRA III (DESY, Hamburg, Germany) using a Pilatus 2M detector at a sample-detector distance of 3.1 m and at a wavelength of λ = 0.124 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). One solute concentration of 1.70 mg/ml was measured at 20°C. 18 successive 0.050 second frames were collected. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted.

EcoKMcrA (as monomeric protein chain) ratio to DNA oligoduplex in the sample was 1:1. NOTE! The DNA used in the models are homologous, and do not necessarily correspond in sequence, to the deposited DNA sequence of the complex analysed using SAXS.

cognate hemimethylated 12-bp oligoduplex (12_5mC/12_C)
Mol. type   DNA
Olig. state   Dimer
Mon. MW   7.4 kDa
Sequence   FASTA
 
5-methylcytosine-specific restriction enzyme A (EcoKMcrA)
Mol. type   Protein
Organism   Escherichia coli K-12
Olig. state   Dimer
Mon. MW   32.3 kDa
 
UniProt   P24200 (1-277)
Sequence   FASTA